(a) Field of the Invention
The invention relates to compositions and methods to treat fungal infection. More particularly, the invention relates to compositions and methods for enhancing fungal sensitivity to antifungal compounds.
(b) Description of Prior Art
In eukaryotic cells, nuclear DNA associates with histones to form a compact complex called chromatin. The histones constitute a family of basic proteins which are generally highly conserved across eukaryotic species. The core histones, termed H2A, H2B, H3, and H4, associate to form a protein core. DNA winds around this protein core, with the basic amino acids of the histones interacting with the negatively charged phosphate groups of the DNA. Approximately 146 base pairs of DNA wrap around a histone core to make up a nucleosome particle, the repeating structural motif of chromatin.
Csordas, (1990, Biochem. J., 286: 23-38) teaches that histones are subject to post-translational acetylation of amino groups of N-terminal lysine residues, a reaction that is catalyzed by histone acetyl transferase (HAT1). Acetylation neutralizes the positive charge of the lysine side chain, and is thought to impact chromatin structure. Indeed, Taunton et al., (1996, Science, 272: 408-411), teaches that access of transcription factors to chromatin templates is enhanced by histone hyperacetylation. Taunton et al. (supra) further teach that an enrichment in under-acetylated histone H4 has been found in transcriptionally silent regions of the genome.
Histone acetylation is a reversible modification, with deacetylation being catalyzed by a family of enzymes termed histone deacetylases (HDACs). The molecular cloning of gene sequences encoding proteins with HDAC activity has established the existence of a set of discrete HDAC enzyme isoforms. Based on phylogenetic analyses and sequence homology to yeast Rpd3 (reduced potassium dependency 3), Hda1 and Sir2 (silent information regulator 2), HDACs are grouped into different classes (Jang and Grégoire, 2005, Molecular and Cellular Biology, 25(8):2873-2884). In humans there are 18 known HDACs, which are divided into four classes: class I (HDAC1, -2, -3 and -8; homologous to Rpd3), class II (HDAC4, -5, -6, -7, -9 and -10; related to Hda1), class III (Sirt1, -2, -3, -4, -5, -6 and -7; similar to Sir2) and class IV (HDAC11). Class I, II and IV HDACs are zinc-dependent enzymes. Class III HDACs are NAD+ dependent deacetylases. In Saccharomyces cerevisiae there are 10 known HDACs, which are divided into three classes: class I (Rpd3, Hos1 and Hos2), class II (Hda1 and Hos3), and class III (Sir2 and four Hst proteins, homologs of Sir2).
It has been unclear what roles these individual HDAC enzymes play. Trojer et al. (2003, Nucleic Acids Research, 31(14):3971-3981) indicate that HdaA and RpdA are major contributors to total HDAC activity of the filamentous fungus Aspergillus nidulans, with HdaA accounting for the main part of the HDAC activity.
Studies utilizing known HDAC inhibitors have established a link between acetylation and gene expression. Numerous studies have examined the relationship between HDAC and gene expression. Taunton et al., Science 272:408-411 (1996), discloses a human HDAC that is related to a yeast transcriptional regulator. Cress et al., J. Cell. Phys. 184:1-16 (2000), discloses that, in the context of human cancer, the role of HDAC is as a corepressor of transcription. Ng et al., TIBS 25: March (2000), discloses HDAC as a pervasive feature of transcriptional repressor systems. Magnaghi-Jaulin et al., Prog. Cell Cycle Res. 4:41-47 (2000), discloses HDAC as a transcriptional co-regulator important for cell cycle progression.
Numerous reports have been made describing inhibitors of HDAC activity. For example, Richon et al., Proc. Natl. Acad. Sci. USA, 95: 3003-3007 (1998), discloses that HDAC activity is inhibited by trichostatin A (TSA), a natural product isolated from Streptomyces hygroscopicus, which has been shown to inhibit histone deacetylase activity and arrest cell cycle progression in cells in the G1 and G2 phases (Yoshida et al., 1990, J. Biol. Chem. 265: 17174-17179; Yoshida et al., 1988, Exp. Cell Res. 177: 122-131), and by a synthetic compound, suberoylanilide hydroxamic acid (SAHA). Yoshida and Beppu (1988, Exper. Cell Res., 177: 122-131) teach that TSA causes arrest of rat fibroblasts at the G1 and G2 phases of the cell cycle, implicating HDAC in cell cycle regulation. Indeed, Finnin et al. (1999, Nature, 401:188-193), teach that TSA and SAHA inhibit cell growth, induce terminal differentiation, and prevent the formation of tumors in mice. Other non-limiting examples of compounds that serve as HDAC inhibitors include those of WO 01/38322 and WO 01/70675. The A. nidulans Hda1 enzyme is highly sensitive to the HDAC inhibitor TSA, while HosB has been shown to be highly resistant to both TSA and another HDAC inhibitor, HC toxin (Trojer et al., supra).
Smith and Edlind (2002, Antimicrobial Agents and Chemotherapy, 46(11):3532-3539) tested the ability of known HDAC pan-inhibitors TSA, apicidin, sodium butyrate and trapoxin to enhance the sensitivity of selected fungal species to azole antifungal agents. They found that only TSA was able to enhance the sensitivity of Candida albicans. However, the concentrations of TSA required were higher than those toxic to mammalian cells. TSA was not found to enhance the sensitivity of Candida glabrata. 
The use of, and need for, antifungal agents is widespread and ranges from the treatment of mycotic infections in animals; to disinfectant formulations; to pharmaceuticals for human use. A major problem with current antifungal formulations is their toxicity to the infected host. This is particularly important in cases where many fungal infestations are opportunistic infections secondary to debilitating diseases, such as AIDS or from cancer chemotherapy or organ transplants. Correspondingly, at least for antifungal agents that are to be administered to humans and other animals, the therapeutic index is preferably such that toxicity is selective to the targeted fungus without being toxic to the host.
Serious fungal infections, caused mostly by opportunistic species such as Candida spp. And Aspergillus spp., are increasingly common in immunocompromised and other vulnerable patients (Georgopapadakou, 1998). They are important causes of morbidity and mortality in hospitalized patients and in HIV, cancer and transplant patients.
Infections by Candida are commonly treated with antifungal azoles which target lanosterol demethylase, an essential enzyme in ergosterol synthesis, the major component of the fungal membrane. Azoles are fungistatic and their use may be eroded by the emergence of azole-resistance, particularly in non-albicans Candida species such as Candida glabrata (Kaur et al., 2004). Further, azole treatment results in “trailing growth”, with surviving fungal cells becoming reservoirs for relapse. The major limitation of antifungal azoles is their general lack of fungicidal activity, which may contribute to treatment failures common with severely compromised patients.
Aspergillus fumigatus is the major Aspergillus species causing invasive aspergillosis (IA), a life-threatening disease with a mortality rate of 60-90%, whose incidence has increased dramatically in the past 20 years due to the increasing numbers of immunocompromised patients (Takaia et al., 2005). Current antifungal agents are limited in the treatment of IA by their poor in vivo efficacy and host toxicity (Latge 1999).
Drawbacks to current antifungal agents, such as the azoles, include development of resistance, possible drug-drug interactions and possible toxic liver effects.
An important factor in the resistance to azoles is thought to be the up-regulation of ERG genes that encode enzymes of the ergosterol biosynthetic pathway. Henry et al. demonstrated that exposure to azoles leads to the up-regulation of ERG11, the gene that encodes lanosterol demethylase, in Candida species. In the same study, up-regulation was also seen to occur in the five other ERG genes examined. Similar results were obtained with terbinafine and fenpropimorph, antifungals that act on other steps of the ergosterol pathway (Henry et al., 2000, Antimicrob. Agents Chemother. 44:2693-2700; Song et al., 2004 Antimicrob. Agents Chemother. 48(4):1136-1144).
It would be highly desirable to be provided with compositions and methods to treat fungal infection. It would also be highly desirable to be provided with compositions and methods for enhancing fungal sensitivity to antifungal compounds. Of particular importance, it would be highly desirable to provide such compositions and methods which are selectively toxic to the pathological fungus without being toxic to the host.